Ser/Arg-rich protein-mediated communication between U1 and U2 small nuclear ribonucleoprotein particles

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5' splice site, can positively influence utilization of an upstream 3' splice site via exon definition in both trans- and cis-splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5' splice site on trans-splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5' splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient trans-splicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis-spliced intron. Weakening of the 3' splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between U1 and U2 snRNPs whether this interaction is across introns or exons.     

A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly

Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.     

Purine-rich enhancers function in the AT-AC pre-mRNA splicing pathway and do so independently of intact U1 snRNP.

A rare class of introns in higher eukaryotes is processed by the recently discovered AT-AC spliceosome. AT-AC introns are processed inefficiently in vitro, but the reaction is stimulated by exon-definition interactions involving binding of U1 snRNP to the 5'' splice site of the downstream conventional intron. We report that purine-rich exonic splicing enhancers also strongly stimulate sodium channel AT-AC splicing. Intact U2, U4, or U6 snRNAs are not required for enhancer function or for exon definition. Enhancer function is independent of U1 snRNP, showing that splicing stimulation by a downstream 5'' splice site and by an exonic enhancer differ mechanistically.     

The intronic splicing code: multiple factors involved in ATM pseudoexon definition

Abundance of pseudo splice sites in introns can potentially give rise to innumerable pseudoexons, outnumbering the real ones. Nonetheless, these are efficiently ignored by the splicing machinery, a process yet to be understood completely. Although numerous 5′ splice site‐like sequences functioning as splicing silencers have been found to be enriched in predicted human pseudoexons, the lack of active pseudoexons pose a fundamental challenge to how these U1snRNP‐binding sites function in splicing inhibition. Here, we address this issue by focusing on a previously described pathological ATM pseudoexon whose inhibition is mediated by U1snRNP binding at intronic splicing processing element (ISPE), composed of a consensus donor splice site. Spliceosomal complex assembly demonstrates inefficient A complex formation when ISPE is intact, implying U1snRNP‐mediated unproductive U2snRNP recruitment. Furthermore, interaction of SF2/ASF with its motif seems to be dependent on RNA structure and U1snRNP interaction. Our results suggest a complex combinatorial interplay of RNA structure and trans‐acting factors in determining the splicing outcome and contribute to understanding the intronic splicing code for the ATM pseudoexon.     

The Pre-mRNA 5′ Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5′ Splice Sites

Efficient splicing of the 5′-most intron of pre-mRNA requires a 5′ m⁷G(5′)ppp(5′)N cap, which has been implicated in U1 snRNP binding to 5′ splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5′ cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5′ splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5′ splice site and not with any loss of U1 snRNP binding.     

Functional recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly

A sensitive assay based on competition between cis-and trans-splicing suggested that factors in addition to U1 snRNP were important for early 5' splice site recognition. Cross-linking and physical protection experiments revealed a functionally important interaction between U4/U6.U5 tri-snRNP and the 5' splice site, which unexpectedly was not dependent upon prior binding of U2 snRNP to the branch point. The early 5' splice site/tri-snRNP interaction requires ATP, occurs in both nematode and HeLa cell extracts, and involves sequence-specific interactions between the highly conserved splicing factor Prp8 and the 5' splice site. We propose that U1 and U5 snRNPs functionally collaborate to recognize and define the 5' splice site prior to establishment of communication with the 3' splice site.     

G Run-mediated recognition of proteolipid protein and DM20 5' splice sites by U1 small nuclear RNA is regulated by context and proximity to the splice site

Highly conserved G runs, G1M2 and ISE, regulate the proteolipid protein (PLP)/DM20 ratio. We have investigated recruitment of U1 small nuclear ribonuclear protein (snRNP) by G1M2 and ISE and examined the effect of splice site strength, distance, and context on G run function. G1M2 is necessary for initial recruitment of U1snRNP to the DM20 5' splice site independent of the strength of the splice site. G1M2 regulates E complex formation and supports DM20 splicing when functional U1snRNP is reduced. By contrast, the ISE is not required for the initial recruitment of U1snRNP to the PLP 5' splice site. However, in close proximity to either the DM20 or the PLP 5' splice site, the ISE recruits U1snRNP to both splice sites. The ISE enhances DM20 splicing, whereas close to the PLP 5' splice site, it inhibits PLP splicing. Splicing enhancement and inhibition are mediated by heterogeneous nuclear ribonuclear protein (hnRNP)H/F. The data show that recognition of the DM20 5' splice site depends on G run-mediated recruitment of U1snRNA, whereas a complex interaction between the ISE G runs, context and position determines the functional outcome on splicing. The data suggest that different mechanisms underlie G run-mediated recognition of 5' splice sites and that context and position play a critical role.     

U11 snRNA interacts in vivo with the 5' splice site of U12-dependent (AU-AC) pre-mRNA introns.

A notable feature of the newly described U12 snRNA-dependent class of eukaryotic nuclear pre-mRNA introns is the highly conserved 8-nt 5'' splice site sequence. This sequence is virtually invariant in all known members of this class from plants to mammals. Based on sequence complementarity between this sequence and the 5'' end of the U11 snRNA, we proposed that U11 snRNP may play a role in identifying and/or activating the 5'' splice site for splicing. Here we show that mutations of the conserved 5'' splice site sequence of a U12-dependent intron severely reduce correct splicing in vivo and that compensatory mutations in U11 snRNA can suppress the effects of the 5'' splice site mutations to varying extents. This provides evidence for a required interaction between U11 snRNA and the 5'' splice site sequence involving Watson-Crick base pairing. This data, in addition to a report that U11 snRNP is bound transiently to the U12-dependent spliceosome, suggests that U11 snRNP is the analogue of U1 snRNP in splicing this rare class of introns.     

Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF.

We have used protection against ribonuclease H to investigate the mechanisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5' splice sites. The initial binding of U1 snRNPs to alternative consensus splice sites was indiscriminate, and on a high proportion of pre-mRNA molecules both sites were occupied simultaneously. When the sites were close, this inhibited splicing. We propose that double occupancy leads to the use of the downstream site for splicing and that this is the cause of the proximity effect seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity of U1 snRNPs for the downstream site. This prediction was tested both by cleaving the 5' end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstream 5' splice site use by splicing factor SF2/ASF appears to be mediated by an increase in the strength of U1 snRNP binding to all sites indiscriminately.     

A role for the Psi-U mismatch in the recognition of the 5' splice site of yeast introns by the U1 small nuclear ribonucleoprotein particle

The U1 small nuclear ribonucleoprotein particle (snRNP)/5' splice site (5'SS) interaction in yeast is essential for the splicing process and depends on the formation of a short RNA duplex between the 5' arm of U1 snRNA and the 1st intronic nucleotides. This RNA/RNA interaction is characterized by the presence of a mismatch that occurs with almost all yeast introns and concerns nucleotides 4 on the pre-mRNA (a U) and 5 on U1 snRNA (a Psi). The latter nucleotide is well conserved from yeast to vertebrates, but its role in yeast and the significance of the associated mismatch in the U1 snRNA/5'SS interaction have never been fully explained. We report here that the presence of this mismatch is a determinant of stability that mainly affects the off rate of the interaction. To our knowledge this is the first report assigning a function to this noncanonical interaction. We also performed SELEX (systematic evolution of ligands by exponential enrichment) experiments by immunoprecipitating U1 snRNP and the associated RNA. The artificial phylogeny derived from these experiments allows the isolation of the selective pressure due to U1 snRNP binding on the 5'SS of yeast introns.     

Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing

Although both U1 and U2 snRNPs have been implicated in the splicing process, their respective roles in the earliest stages of intron recognition and spliceosome assembly are uncertain. To address this issue, we developed a new strategy to prepare snRNP-depleted splicing extracts using Saccharomyces cerevisiae cells conditionally expressing U1 or U2 snRNP. Complementation analyses and chase experiments show that a stable complex, committed to the splicing pathway, forms in the absence of U2 snRNP. U1 snRNP and a substrate containing both a 5' splice site and a branchpoint sequence are required for optimal formation of this commitment complex. We developed new gel electrophoresis conditions to identify these committed complexes and to show that they contain U1 snRNA. Chase experiments demonstrated that these complexes are functional intermediates in spliceosome assembly and splicing. Our results have implications for the process of splice site selection.     

A downstream splicing enhancer is essential for in vitro pre-mRNA splicing.

Splicing enhancers have previously been shown to promote processing of introns containing weak splicing signals. Here, we extend these studies by showing that also 'strong' constitutively active introns are absolutely dependent on a downstream splicing enhancer for activity in vitro. SR protein binding to exonic enhancer elements or U1 snRNP binding to a downstream 5' splice site serve redundant functions as activators of splicing. We further show that a 5' splice site is most effective as an enhancer of splicing. Thus, a 5' splice site is functional in S100 extracts, under conditions where a SR enhancer is nonfunctional. Also, splice site pairing occurs efficiently in the absence of exonic SR enhancers, emphasizing the significance of a downstream 5' splice site as the enhancer element in vertebrate splicing.     

Mer1p is a modular splicing factor whose function depends on the conserved U2 snRNP protein Snu17p

Mer1p activates the splicing of at least three pre-mRNAs (AMA1, MER2, MER3) during meiosis in the yeast Saccharomyces cerevisiae. We demonstrate that enhancer recognition by Mer1p is separable from Mer1p splicing activation. The C-terminal KH-type RNA-binding domain of Mer1p recognizes introns that contain the Mer1p splicing enhancer, while the N-terminal domain interacts with the spliceosome and activates splicing. Prior studies have implicated the U1 snRNP and recognition of the 5′ splice site as key elements in Mer1p-activated splicing. We provide new evidence that Mer1p may also function at later steps of spliceosome assembly. First, Mer1p can activate splicing of introns that have mutated branch point sequences. Secondly, Mer1p fails to activate splicing in the absence of the non-essential U2 snRNP protein Snu17p. Thirdly, Mer1p interacts with the branch point binding proteins Mud2p and Bbp1p and the U2 snRNP protein Prp11p by two-hybrid assays. We conclude that Mer1p is a modular splicing regulator that can activate splicing at several early steps of spliceosome assembly and depends on the activities of both U1 and U2 snRNP proteins to activate splicing.     

3' splice site recognition in nematode trans-splicing involves enhancer-dependent recruitment of U2 snRNP.

Trans-splicing requires that 5' and 3' splice sites be independently recognized. Here, we have used mutational analyses and a sensitive nuclease protection assay to determine the mechanism of trans-3' splice site recognition in vitro. Efficient recognition of the 3' splice site is dependent upon both the sequence of the 3' splice site itself and enhancer elements located in the 3' exon. We show that the presence of three distinct classes of enhancers results in increased binding of U2 snRNP to the branchpoint region. Several lines of evidence strongly suggest that the increased binding of U2 snRNP is mediated by U2AF. These results expand the roles of enhancers in constitutive splicing and provide direct support for the recruitment model of enhancer function.     

A novel role of U1 snRNP: Splice site selection from a distance

Removal of introns by pre-mRNA splicing is fundamental to gene function in eukaryotes. However, understanding the mechanism by which exon-intron boundaries are defined remains a challenging endeavor. Published reports support that the recruitment of U1 snRNP at the 5′ss marked by GU dinucleotides defines the 5′ss as well as facilitates 3′ss recognition through cross-exon interactions. However, exceptions to this rule exist as U1 snRNP recruited away from the 5′ss retains the capability to define the splice site, where the cleavage takes place. Independent reports employing exon 7 of Survival Motor Neuron (SMN) genes suggest a long-distance effect of U1 snRNP on splice site selection upon U1 snRNP recruitment at target sequences with or without GU dinucleotides. These findings underscore that sequences distinct from the 5′ss may also impact exon definition if U1 snRNP is recruited to them through partial complementarity with the U1 snRNA. In this review we discuss the expanded role of U1 snRNP in splice-site selection due to U1 ability to be recruited at more sites than predicted solely based on GU dinucleotides.     

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).     

U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered.